Abstract A key virulence attribute of any microbial pathogen is the ability to adapt to the conditions of the infected host. One of these host-associated conditions is neutral/alkaline extracellular pH. Fungal pathogens use the Rim/Pal signal transduction pathway to sense and respond to host pH. Mutants in this pathway are attenuated for survival at alkaline pH and in the infected host. The human fungal pathogen, Cryptococcus neoformans is an important cause of opportunistic infections in immunocompromised patients. This pathogen uses the Rim pathway to regulate cellular changes necessary for growth at alkaline pH. This pathway was first described in the ascomycete phylum. Interestingly, C. neoformans, a basidiomycete, lacks genes encoding many of the components of the Rim pathway, including much of the membrane pH-sensing complex. This proposal aims to identify proteins in the C. neoformans Rim pathway that sense changes in pH and investigate how C. neoformans and related basidiomycetes use different strategies for sensing extracellular pH compared to model ascomycete fungi. In Aim 1, I will use an insertional mutagenesis screen to identify. They will be further prioritized based on phylum specificity and compelling secondary structures. In Aim 2, I will pursue an interesting finding from my genetic screen in Aim 1 ? that mutants with plasma membrane composition defects are unable to grow at alkaline pH. In this aim, I will specifically focus on membrane bilayer asymmetry as a dynamic cellular trigger of altered extracellular pH. I will test if the C. neoformans Cdc50 flippase, and maintenance of plasma membrane asymmetry, is required for Rim pathway activation. These results will elucidate the molecular interactions that drive environment-sensing in a large and biologically diverse group of fungi.